Direct analysis of crude plasma samples by turbulent flow chromatography/tandem mass spectrometry

Abstract

Turbulent flow chromatography coupled to tandem mass spectrometry (TFC-MS-MS) has recently emerged as a potentially fast, sensitive and specific technique for the direct analysis of pharmaceutical compounds from crude plasma. TFC-MS-MS removes the need for time-consuming sample preparation procedures such as solid-phase extraction (SPE) or liquid-liquid extraction (LLE). A relatively high flow rate combined with the use, of an HPLC column with large porous particles allows the on-line clean up and quantification of compounds in plasma samples. Until, now, the amount of plasma directly injected into TFC systems has rarely exceeded 30 μL in order to prevent rapid column degradation. Increasing the injection volume also induces high carry-over levels, particularly for drugs with basic and/or lipophilic properties. This paper describes the first genetic TFC-MS-MS method developed in a 96-well format, which allows the direct injection of 200 μL of 1∶1 diluted plasma (equivalent to 100 μL neat plasma). An average, of 390 injections was carried out with each extraction column. More than 2000 dog plasma samples were injected into the system without any sign of carryover. The method was fully validated over a 5–500 ng mL−1 range for three basic compounds: doxazosin, CP122,288 and dofetilide. The imprecision was 1.2 to 8.3% for doxazosin, 1.5 to 4% for CP122,288 and 1.6 to 9.2% for dofetilide. The inaccuracy ranged from 6% to 7.9%. This generic methodology was then used to assay two structurally unrelated development compounds, showing that the method accuracy and sensitivity were adequate for the early pharmacokinetic (PK) studies in animals.